Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: An environmental- and cryo-scanning electron microscopy study Fabio Nudelman, Eyal Shimoni, Eugenia Klein, Marthe Rousseau, Xavier Bourrat, Evelyne Lopez, Lia Addadi, Steve Weiner To cite this version: Fabio Nudelman, Eyal Shimoni, Eugenia Klein, Marthe Rousseau, Xavier Bourrat, et al.. Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: An environmental- and cryo-scanning electron microscopy study. Journal of Structural Biology, Elsevier, 2008, 162, pp.290-300. 10.1016/j.jsb.2008.01.008. insu-00287022 HAL Id: insu-00287022 https://hal-insu.archives-ouvertes.fr/insu-00287022 Submitted on 10 Jun 2008 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: An environmental- and cryo-scanning electron microscopy study Fabio Nudelman a, Eyal Shimonib, Eugenia Kleinb, Marthe Rousseauc, Xavier Bourratd, Evelyne Lopezc, Lia Addadia and Steve Weinera aDepartment of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel bDepartment of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel cMuséum National d’Histoire Naturelle, Départment des Milieux et Peuplements Aquatiques UMR 5178 : CNRS-MNHN : Biologie des Organismes Marins et Ecosystemes 43, rue Cuvier CP26, 75005 Paris, France dInstitut des Sciences de la Terre d’Orleans, CNRS-Université d’Orleans, 1A, rue de la Ferollerie, 45071 Orleans cedex 2, France Abstract A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer. Keywords: Biomineralization; Mollusk shell nacre; Matrix macromolecules; Cryo-scanning electron microscopy; Silk fibroin 1. Introduction The nacreous layer, or “mother-of-pearl”, is the innermost layer of many mollusk shells. It is widely studied as a model for understanding biomineralization processes, because of its regular brick wall-like structure. It is composed of polygonal aragonite crystals that are 5– 15 μm in diameter. They are arranged in continuous parallel laminae around 0.5 μm thick, separated by sheets of interlamellar organic matrix ([Gregoire, 1957], [Watabe, 1965] and [Wise, 1970]). Nacre tablets are single crystals with a sub-structure composed of 30–50 nm domains embedded in organic material (Rousseau et al., 2005b). TEM studies of the growing nacre have demonstrated that the interlamellar matrix is formed prior to mineral deposition (Bevelander and Nakahara, 1969) and that in the mature nacre it is composed of an electron lucent layer enclosed by two electron dense layers (Nakahara, 1979). The major component of the interlamellar matrix is β-chitin (Levi-Kalisman et al., 2001), whose fibrils are preferably aligned in a direction parallel to the a-axis of the juxtaposed crystals, indicating that the crystal orientation upon nucleation is governed, either directly or indirectly, by chitin (Weiner and Traub, 1984). Levi-Kalisman et al. (2001) were not able to image the other major components of the matrix, the silk-like proteins ([Weiner et al., 1983] and [Weiner and Traub, 1980]), and suggested that they are present in a hydrated gel-like state filling the space between two layers of chitin prior to mineral formation. Although preliminary results supporting this hypothesis were provided from experiments on mature shells (Addadi et al., 2006), the involvement of a gel phase in the forming stages still remains to be demonstrated. An assemblage of relatively soluble macromolecules forms a third class of macromolecules. Many of these are glycoproteins rich in aspartic acid ([Crenshaw, 1972] and [Weiner, 1979]). Some of these proteins are adsorbed on the chitin scaffold and constitute the nucleation site of the forming tablet ([Crenshaw and Ristedt, 1976] and [Nudelman et al., 2006]). In addition, some are able to specifically nucleate aragonite ([Belcher et al., 1996] and [Falini et al., 1996]). Another group of the aspartic acid-rich proteins are located within the mineral phase ([Berman et al., 1993] and [Weiner and Addadi, 1997]). Although β-chitin, the silk-like proteins and the acidic proteins are considered the major components of the organic matrix of the nacre, several other proteins have been identified and characterized over the past 10 years for a review on mollusk shell proteins see Marin and Luquet (2004). The zone between the mantle and the shell is of much importance for the understanding of shell formation. This zone is filled with the extrapallial fluid, into which it is often assumed that the matrix macromolecules and the ions necessary for mineralization are secreted by the mantle cells (Bevelander and Nakahara, 1969). It has been subsequently surmised that the macromolecules self-assemble in the extrapallial fluid to form the organic matrix and later mineralization occurs. It is more likely, however, that the mantle cells are juxtaposed to the mineralizing matrix where and when shell is produced (Simkiss and Wilbur, 1989). Furthermore, a viscous fluid in the space where the nacre tablets form between the mantle cells and the nacre surface, was reported (Rousseau et al., 2005a). A fibrous thin organic film adhering to the growing mineral surface was also observed in some mollusk species, and in many cases it contained associated particles and possibly crystals ([Neff, 1972] and [Waller, 1980]). In the shell of Mercenaria mercenaria, this layer is observed immediately above the microvilli of the mantle epithelium (Neff, 1972). The composition of this film and its functional significance are not known. The mechanisms of mineral transport to the mineralization site are also an important issue in shell formation. Observations of mineral-containing granules within the mantle epithelial cells suggest that calcium carbonate is not transported in solution, but as an already formed mineral phase (Neff, 1972). The nature of the first deposited mineral phase in nacre—whether crystalline or not—is not clear. Weiss et al. (2002) showed that in mollusk larvae, the first- formed mineral phase is amorphous calcium carbonate (ACC), which subsequently transforms into aragonite. Echinoderms and sponges are also known to use an ACC precursor phase to form their skeletons and spicules ([Politi et al., 2004] and [Sethmann et al., 2006]). Although it is not known whether the same strategy is used in nacre formation, there is some indirect evidence that this might be the case. The mineral containing granules observed by Neff (1972) did not produce electron diffraction patterns, and therefore could be composed of ACC. Nassif et al. (2005) have shown that nacreous tablets of adult mollusk shells are coated by a thin layer of ACC, suggesting that this phase is present in nacre. The microenvironment formed by the 3-dimensional organic matrix framework is critical for proper regulation over mineral formation. Much of what is known to date about nacre formation comes from studies of the mature tissue. Although these studies have been extremely important, this approach is rather limited as it involves investigating the fully formed layer in order to understand its process of formation. In particular, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Hence, the study of a mineralized tissue during its formation process is essential. This however is not trivial, and requires the use of appropriate methodologies. Here, we employed environmental scanning electron microscopy (ESEM) and cryo-scanning electron microscopy (cryo-SEM) in order to investigate the space between two interlamellar sheets, namely the site where the mineral forms, in